PROJECT SUMMARY/ABSTRACT Atrial fibrillation (afib) is a common arrhythmia that affects more than 3 million people in the United States and contributes to substantial morbidity and mortality. The goals of medical therapy for all patients are to maintain sinus rhythm and to avoid the risk of complications (especially the increased risk of stroke) while minimizing symptoms to provide an acceptable quality of life. Current antiarrhythmic drugs are plagued by significant adverse side effects and a relative lack of effectiveness. For example, the most effective drug for the maintenance of sinus rhythm in afib patients is amiodarone - a drug well known to have a significant number of off-target side effects. Despite its superior efficacy, the use of amiodarone is currently limited because its systemic method of delivery leads to toxicity in lung, liver, thyroid, and ocular tissue when it is administered long-term. The goal of our project is to ameliorate the off-target adverse effects of amiodarone (AM) by conjugating it to our proprietary cell penetrating peptide, Cardiac Targeting Peptide (CTP) to create a CTP-AM conjugate that will deliver this drug specifically and directly to the heart. CTP is at the center of the technical innovation outlined in this grant proposal. It is a novel, synthetic peptide that has demonstrated ability to transduce (i) normal mouse hearts in vivo (peak uptake at 15 minutes after injection), (ii) explanted human heart tissue, and (iii) human derived iPSC beating cardiomyocytes. CTP demonstrates robust transduction of normal cardiomyocytes with sparing of fibroblasts present in adjacent scar tissue. To date, CTP conjugates have transduced cardiomyocytes carrying intact cargoes as diverse as nucleic acids, therapeutics, radio-isotopes, other peptides, and conjugates as large as the biotin-streptavidin conjugate. We believe CTP is fully capable of carrying AM in a similar manner. Our hypothesis is that CTP-AM will transduce normal cardiomyocytes, delivering the drug where it is needed to act, while significantly reducing the off-target toxicities associated with its current chronic administration. To test this hypothesis, we aim to 1) optimize a stable conjugate of amiodarone to CTP; 2) test the transduction efficiency, stability, anti-arrhythmic efficacy, and dose responsiveness of the optimized conjugate in guinea pig hearts ex vivo and 3) test transduction efficiencies and anti-arrhythmic efficacy of CTP-AM, against CTP alone and AM alone in vivo in guinea pigs. Our Phase II objectives will be to run pre-clinical trials of AM vs. CTP-AM conjugates to suppress afib. We will use two afib models: a) spontaneously hypertensive (1-year old) and b) aged (~2 year old) rats that develop long lasting afib. We expect that CTP-AM conjugates will suppress afib at ~1/10th the concentration of AM alone and will avoid off-target toxic effects. These studies will form the basis and rationale for conducting full pharmaco-toxicity studies on CTP-AM to support an Investigational New Drug (IND) application to the FDA for a Phase I clinical trial with CTP-AM.